Growth differentiation factor-16

ABSTRACT

Growth differentiation factor-16 (GDF-16) is disclosed along with its polynucleotide sequence and amino acid sequence Also disclosed are diganostic and therapeutic methods of using the GDF-16 polypeptide and polynucleotide sequences.

CROSS REFERENCE TO RELATED APPLICATIONS

Under 35 USC § 120, this application is a continuation application ofU.S. application Ser. No. 09/485,045 filed May 12, 2000, now issued asU.S. Pat. No. 7,098,323; which is a 35 USC § 371 National Stageapplication of PCT Application No. PCT/US98/15148 filed Jul. 24, 1998;which claims the benefit under 35 USC § 119(e) to U.S. application Ser.No. 60/054,606 filed Jul. 31, 1997, now abandoned. The disclosure ofeach of the prior applications is considered part of and is incorporatedby reference in the disclosure of this application.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to growth factors and specifically to anew member of the transforming growth factor beta (TGF-β) superfamily,which is denoted, growth differentiation factor-16 (GDF-16).

2. Description of Related Art

The transforming growth factor β (TGF-β) superfamily encompasses a groupof structurally-related proteins which affect a wide range ofdifferentiation processes during embryonic development. The familyincludes, Mullerian inhibiting substance (MIS), which is required fornormal male sex development (Behringer. et al., Nature, 345:167, 1990),Drosophila decapentaplegic (DPP) gene product, which is required fordorsal-ventral axis formation and morphogenesis of the imaginal disks(Padgett, et al., Nature, 325:81-84, 1987), the Xenopus Vg-1 geneproduct, which localizes to the vegetal pole of eggs (Weeks, et al.Cell, 51:861-867, 1987), the activins (Mason, et al., Biochem, Biophys.Res. Commun., 135:957-964. 1986), which can induce the formation ofmesoderm and anterior structures in Xenopus embryos (Thomsen, et al.,Cell, 63:485, 1990), GDNF, which can promote the survival of motorneurons and midbrain dopaminergic neurons (Lin, et al., Science,260:1130, 1993; Tomae. et al., Nature, 373:335,1995; Beck, et al.,Nature, 373:339, 1995; Henderson, el al. Science, 266:1062, 1994; Van,et al., Nature. 373:341.1995; Oppenheim, et al., Nature, 373:344, 1995)and the bone morphogenetic proteins (BMPs, osteogenin, OP-1) which caninduce de novo cartilage and bone formation (Sampath, et al., J Biol.Chem., 265:13198, 1990). The TGF-βs can influence a variety ofdifferentiation processes, including adipogenesis, myogenesis,chondrogenesis, hematopoiesis, and epithelial cell differentiation (forreview, see Massague, Cell 49:437, 1987).

The proteins of the TGF-β family are initially synthesized as a largeprecursor protein which subsequently undergoes proteolytic cleavage at acluster of basic residues approximately 110-140 amino acids from theC-terminus. The C-terminal regions, or mature regions, of the proteinsare all structurally related and the different family members can beclassified into distinct subgroups based on the extent of theirhomology. Although the homologies within particular subgroups range from70% to 90% amino acid sequence identity, the homologies betweensubgroups are significantly lower, generally ranging from only 20% to50%. In each case, the active species appears to be a disulfide-linkeddimer of C-terminal fragments. Studies have shown that when thepro-region of a member of the TGF-β family is coexpressed with a matureregion of another member of the TGF-β family, intracellular dimerizationand secretion of biologically active homodimers occur (Gray, A., andMaston, A., Science, 247:1328, 1990). Additional studies by Hammonds, etal., (Molec. Endochn. 5:149,1991) showed that the use of the BMP-2pro-region combined with the BMP-4 mature region led to dramaticallyimproved expression of mature BMP-4. For most of the family members thathave been studied, the homodimeric species has been found to bebiologically active, but for other family members, like the inhibins(Ling, et al., Nature, 321:779,1986) and the TGF-βs (Cheifetz, et al,Cell, 48:409, 1987), heterodimers have also been detected, and theseappear to have different biological properties than the respectivehomodimers.

Identification of new factors that are tissue-specific in theirexpression pattern will provide a greater understanding of that tissue'sdevelopment and function.

SUMMARY OF THE INVENTION

The present invention provides a cell growth and differentiation factor,GDF-16, a polynucleotide sequence which encodes the factor, andantibodies which are bind to the factor. This factor appears to relateto various cell proliferative disorders.

Thus, in one embodiment, the invention provides a method for detecting acell proliferative disorder which is associated with GDF-16. In anotherembodiment, the invention provides a method for treating a cellproliferative or immunologic disorder by suppressing or enhancing GDF-16activity.

In another embodiment, the invention provides a method for identifyingGDF-16 receptor polypeptide comprising incubating components comprisingGDF-16 polypeptide and a cell expressing a receptor or a solublereceptor under conditions sufficient to allow the GDF to bind to thereceptor; measuring the binding of the GDF polypeptide to the receptor;and isolating the receptor. Methods of isolating the receptors aredescribed in more detail in the Examples section.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide and predicted amino acid sequence of humanGDF-16.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a growth and differentiation factor,GDF-16, and a polynucleotide sequence encoding GDF-16. In oneembodiment, the invention provides a method for detection of a cellproliferative or immunologic disorder which is associated with GDF-16expression or function. In another embodiment, the invention provides amethod for treating a cell proliferative or immunologic disorder byusing an agent which suppresses or enhances GDF-16 activity.

The TGF-β superfamily consists of multifunctional polypeptides thatcontrol proliferation, differentiation, and other functions in many celltypes. Many of the peptides have regulatory, both positive and negative,effects on other peptide growth factors. The structural homology betweenthe GDF-16 protein of this invention and the members of the TGF-βfamily, indicates that GDF-16 is a new member of the family of growthand differentiation factors. Based on the known activities of many ofthe other members, it can be expected that GDF-16 will also possessbiological activities that will make it useful as a diagnostic andtherapeutic reagent.

Certain members of this superfamily have expression patterns or possessactivities that relate to the function of the nervous system. Forexample, one family member, namely GDNF, has been shown to be a potentneurotrophic factor that can promote the survival of dopaminergicneurons (Lin, et al., Science, 260:1130, 1993) and motor neurons(Henderson, et al., Science, 266:1062, 1994; Yan. et al., Nature,373:341, 1995; Oppenheim, et al., Nature, 373:344,1995). Another familymember, namely dorsalin-1, is capable of promoting the differentiationof neural crest cells (Basler, et al., Cell, 73:687, 1993). The inhibinsand activins have been shown to be expressed in the brain (Meunier, etal., Proc. Nat'l Acad. Sci, USA, 85:247, 1988; Sawchenko, et al.,Nature, 334:615, 1988), and activin has been shown to be capable offunctioning as a nerve cell survival molecule (Schubert, et al., Nature,344:868, 1990). Another family member, namely GDF-1, is nervoussystem-specific in its expression pattern (Lee, Proc. Nat'l. Acad. Sci,USA, 88:4250, 1991), and certain other family members, such as Vgr-1(Lyons, et al., Proc. Nat'l. Acad. Sci., USA, 86:4554, 1989; Jones, etal., Development, 111:581, 1991), OP-1 (Ozkaynak, etal., J Biol. Chem.,267:25220, 1992), and BMP-4 (Jones, et al., Development, 11 1:531,1991), are also known to be expressed in the nervous system.

GDF-16 may also have applications in treating disease processesinvolving muscle, such as in musculodegenerative diseases or in tissuerepair due to trauma. In this regard, many other members of the TGF-βfamily are also important mediators of tissue repair. TGF-β has beenshown to have marked effects on the formation of collagen and to cause astriking angiogenic response in the newborn mouse (Roberts, et al.,Proc. Natl Acad. Sci., USA 83:4167, 1986). TGF-β has also been shown toinhibit the differentiation of myoblasts in culture (Massague, et al.,Proc. Natl. Acad. Sci., USA 83:8206, 1986). Moreover, because myoblastcells may be used as a vehicle for delivering genes to muscle for genetherapy, the properties of GDF-16 could be exploited for maintainingcells prior to transplantation or for enhancing the efficiency of thefusion process.

GDF-16 may also have applications in the treatment of immunologicdisorders. In particular, TGF-β has been shown to have a wide range ofimmunoregulatory activities, including potent suppressive effects on Band T cell proliferation and function (for review, see Palladino, etal., Ann. N. Y. Acad. Sci., 593:181, 1990). GDF-16 may possess similaractivities and therefore, may be used as an anti-inflammatory agent oras a treatment for disorders related to abnormal proliferation orfunction of lymphocytes.

GDF-16 may have applications in promoting various types of wound repair.Many of the members of the TGF-β family are known to be importantmediators of tissue repair. TGF-β has been shown to have marked effectson the formation of collagen and causes a striking angiogenic responsein the newborn mouse (Roberts, et al., Proc. Natl. Acad. Sci. USA,83:4167, 1986). The BMP's can induce new cartilage and bone growth andare effective for the treatment of fractures and other skeletal defects(Glowacki, et al., Lancet, 1:959, 1981; Ferguson, et al., Clin.Orthoped. Relat. Res., 227:265, 1988; Johnson, et al., Clin. Orthoped.Relat. Res., 230:257, 1988). GDF-16 may have similar or relatedactivities and may be useful in repair of tissue injury caused by traumaor burns, for example.

GDF-16 may play a role in regulating various aspects of fertility ormaternal or fetal growth or function during pregnancy. Hence, GDF-16 oragents that interfere with GDF-16 function may be useful incontraceptive regimens, in enhancing the success of in vitrofertilization procedures, in preventing premature labor, or in enhancingfetal growth or development.

GDF-16 may also have applications in the treatment of various types ofcancer. Several known members of this family can function as tumorsuppressors. For example, inhibin alpha has been shown to suppress thedevelopment of both gonadal (Matzuk, et al., Nature, 360:313, 1992) andadrenal (Matzuk, et al., PNAS, 91:8817, 1984) tumors. Similarly, MIS hasbeen shown to inhibit the growth of human endometrial and ovarian tumorsin nude mice (Donahoe, et al., Ann. Surg., 194:472, 1981). Finally,TGF-β is a potent growth inhibitor for many cell types, and resistanceto TGF-β plays an important role in the progression of colon cancer(Markowitz, et al., Science, 268:1336, 1995).

The term “substantially pure” as used herein refers to GDF-16 which issubstantially free of other proteins, lipids, carbohydrates or othermaterials with which it is naturally associated. One skilled in the artcan purify GDF-16 using standard techniques for protein purification.The substantially pure polypeptide will yield a single major band on anon-reducing polyacrylamide gel. The purity of the GDF-16 polypeptidecan also be determined by amino-terminal amino acid sequence analysis.GDF-16 polypeptide includes functional fragments of the polypeptide, aslong as the activity of GDF-16 remains. Smaller peptides containing thebiological activity of GDF-16 are included in the invention.

The invention provides polynucleotides encoding the GDF-16 protein.These polynucleotides include DNA, cDNA and RNA sequences which encodeGDF-16. It is understood that all polynucleotides encoding all or aportion of GDF-16 are also included herein, as long as they encode apolypeptide with GDF-16 activity. Such polynucleotides include naturallyoccurring, synthetic, and intentionally manipulated polynucleotides. Forexample, GDF-16 polynucleotide may be subjected to site-directedmutagenesis. The polynucleotide sequence for GDF-16 also includesantisense sequences. The polynucleotides of the invention includesequences that are degenerate as a result of the genetic code. There are20 natural amino acids, most of which are specified by more than onecodon. Therefore, all degenerate nucleotide sequences are included inthe invention as long as the amino acid sequence of GDF-16 polypeptideencoded by the nucleotide sequence is functionally unchanged.

Specifically disclosed herein is a partial DNA sequence containing thehuman GDF-16 gene. The sequence contains an open reading frame encodinga protein showing significant homology to know members of the TGF-βsuperfamily. The sequence contains a putative RXXR proteolyticprocessing site that is followed by a sequence of 101 amino acids thatcontains all of the hallmarks of the TGF-β superfamily. By analogy withknown family members, a dimer of the C-terminal 101 amino acids ispredicted to represent the active GDF-16 molecule. Preferably, the humanGDF-16 nucleotide sequence is SEQ ID NO:1 or the nucleotide sequence ofFIG. 1, having 303 nucleotides.

The polynucleotide encoding GDF-16 includes FIG. 1 (SEQ ID NOT), as wellas nucleic acid sequences complementary to SEQ ID NO:1. A complementarysequence may include an antisense nucleotide. When the sequence is RNA,the deoxynucleotides A, G, C, and T of SEQ ID NOT are replaced byribonucleotides A, G, C, and U, respectively. Also included in theinvention are fragments of the above-described nucleic acid sequencesthat are at least 15 bases in length, which is sufficient to permit thefragment to selectively hybridize to DNA that encodes the protein of SEQID NO: 2 under physiological conditions or a close family member ofGDF-16. The term “selectively hybridize” refers to hybridization undermoderately or highly strigent conditions which excludes non-relatednucleotide sequences.

In nucleic acid hybridization reactions, the conditions used to achievea particular level of stringency will vary, depending on the nature ofthe nucleic acids being hybridized. For example, the length, degree ofcomplementarity, nucleotide sequence composition (e.g., GC v. ATcontent), and nucleic acid type (e.g., RNA v. DNA) of the hybridizingregions of the nucleic acids can be considered in selectinghybridization conditions. An additional consideration is whether one ofthe nucleic acids is immobilized, for example, on a filter.

An example of progressively higher stringency conditions is as follows:2×SSC/0.1% SDS at about room temperature (hybridization conditions);0.2×SSC/0.1% SDS at about room temperature (low stringency conditions);0.2×SSC/0.1% SDS at about 42° C. (moderate stringency conditions); and0.1×SSC at about 68° C. (high stringency conditions). Washing can becarried out using only one of these conditions, e.g., high stringencyconditions, or each of the conditions can be used, e.g., for 10-15minutes each, in the order listed above, repeating any or all of thesteps listed. However, as mentioned above, optimal conditions will vary,depending on the particular hybridization reaction involved, and can bedetermined empirically.

The C-terminal region of GDF-16 following the putative proteolyticprocessing site shows significant homology to the known members of theTGF-β superfamily. The GDF-16 sequence contains most of the residuesthat are highly conserved in other family members (see FIG. 1).

Minor modifications of the recombinant GDF-16 primary amino acidsequence may result in proteins which have substantially equivalentactivity as compared to the GDF-16 polypeptide described herein. Suchmodifications may be deliberate, as by site-directed mutagenesis, or maybe spontaneous. All of the polypeptides produced by these modificationsare included herein as long as the biological activity of GDF-16 stillexists. Further, deletion of one or more amino acids can also result ina modification of the structure of the resultant molecule withoutsignificantly altering its biological activity. This can lead to thedevelopment of a smaller active molecule which would have broaderutility. For example, one can remove amino or carboxy terminal aminoacids which are not required for GDF-16 biological activity.

The nucleotide sequence encoding the GDF-16 polypeptide of the inventionincludes the disclosed sequence and conservative variations thereof. Theterm “conservative variation” as used herein denotes the replacement ofan amino acid residue by another, biologically similar residue. Examplesof conservative variations include the substitution of one hydrophobicresidue such as isoleucine, valine, leucine or methionine for another,or the substitution of one polar residue for another, such as thesubstitution of arginine for lysine, glutamic for aspartic acid, orglutamine for asparagine, and the like. The term “conservativevariation” also includes the use of a substituted amino acid in place ofan unsubstituted parent amino acid provided that antibodies raised tothe substituted polypeptide also immunoreact with the unsubstitutedpolypeptide.

DNA sequences of the invention can be obtained by several methods. Forexample, the DNA can be isolated using hybridization techniques whichare well known in the art. These include, but are not limited to: 1)hybridization of genomic or cDNA libraries with probes to detecthomologous nucleotide sequences, 2) polymerase chain reaction (PCR) ongenomic DNA or cDNA using primers capable of annealing to the DNAsequence of interest, and 3) antibody screening of expression librariesto detect cloned DNA fragments with shared structural features.

Preferably the GDF-16 polynucleotide of the invention is derived from amammalian organism, and most preferably from a mouse, rat, or human.Screening procedures which rely on nucleic acid hybridization make itpossible to isolate any gene sequence from any organism, provided theappropriate probe is available. Oligonucleotide probes, which correspondto apart of the sequence encoding the protein in question, can besynthesized chemically. This requires that short, oligopeptide stretchesof amino acid sequence must be known. The DNA sequence encoding theprotein can be deduced from the genetic code, however, the degeneracy ofthe code must be taken into account. It is possible to perform a mixedaddition reaction when the sequence is degenerate. This includes aheterogeneous mixture of denatured double-stranded DNA. For suchscreening, hybridization is preferably performed on eithersingle-stranded DNA or denatured double-stranded DNA. Hybridization isparticularly useful in the detection of cDNA clones derived from sourceswhere an extremely low amount of mRNA sequences relating to thepolypeptide of interest are present. In other words, by using stringenthybridization conditions directed to avoid non-specific binding, it ispossible, for example, to allow the autoradiographic visualization of aspecific cDNA clone by the hybridization of the target DNA to thatsingle probe in the mixture which is its complete complement (Wallace,et al., Nucl. Acid Res., 9:879, 1981; Maniatis, et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor, N.Y. 1989).

The development of specific DNA sequences encoding GDF-16 can also beobtained by: 1) isolation of double-stranded DNA sequences from thegenomic DNA; 2) chemical manufacture of a DNA sequence to provide thenecessary codons for the polypeptide of interest; and 3) in vitrosynthesis of a double-stranded DNA sequence by reverse transcription ofmRNA isolated from a eukaryotic donor cell. In the latter case, adouble-stranded DNA complement of mRNA is eventually formed which isgenerally referred to as cDNA.

Of the three above-noted methods for developing specific DNA sequencesfor use in recombinant procedures, the isolation of genomic DNA isolatesis the least common. This is especially true when it is desirable toobtain the microbial expression of mammalian polypeptides due to thepresence of introns.

The synthesis of DNA sequences is frequently the method of choice whenthe entire sequence of amino acid residues of the desired polypeptideproduct is known. When the entire sequence of amino acid residues of thedesired polypeptide is not known, the direct synthesis of DNA sequencesis not possible and the method of choice is the synthesis of cDNAsequences. Among the standard procedures for isolating cDNA sequences ofinterest is the formation of plasmid- or phage-carrying cDNA librarieswhich are derived from reverse transcription of mRNA which is abundantin donor cells that have a high level of genetic expression. When usedin combination with polymerase chain reaction technology, even rareexpression products can be cloned. In those cases where significantportions of the amino acid sequence of the polypeptide are known, theproduction of labeled single or double-stranded DNA or RNA probesequences duplicating a sequence putatively present in the target cDNAmay be employed in DNA/DNA hybridization procedures which are carriedout on cloned copies of the cDNA which have been denatured into asingle-stranded form (Jay, et al., Nucl. Acid Res., 11:2325, 1983).

A cDNA expression library, such as lambda gtl 1, can be screenedindirectly for GDF-16 peptides having at least one epitope, usingantibodies specific for GDF-16. Such antibodies can be eitherpolyclonally or monoclonally derived and used to detect expressionproduct indicative of the presence of GDF-16 cDNA.

DNA sequences encoding GDF-16 can be expressed in vitro by DNA transferinto a suitable host cell. “Host cells” are cells in which a vector canbe propagated and its DNA expressed. The term also includes any progenyof the subject host cell. It is understood that all progeny may not beidentical to the parental cell since there may be mutations that occurduring replication. However, such progeny are included when the term“host cell” is used. Methods of stable transfer, meaning that theforeign DNA is continuously maintained in the host, are known in theart.

In the present invention, the GDF-16 polynucleotide sequences may beinserted into a recombinant expression vector. The term “recombinantexpression vector” refers to a plasmid, virus or other vehicle known inthe art that has been manipulated by insertion or incorporation of theGDF-16 genetic sequences. Such expression vectors contain a promotersequence which facilitates the efficient transcription of the insertedgenetic sequence of the host. The expression vector typically containsan origin of replication, a promoter, as well as specific genes whichallow phenotypic selection of the transformed cells. Vectors suitablefor use in the present invention include, but are not limited to theT7-based expression vector for expression in bacteria (Rosenberg, etal., Gene, 56:125, 1987), the pMSXND expression vector for expression inmammalian cells (Lee and Nathans, J. Biol. Chem., 263:3521, 1988) andbaculovirus-derived vectors for expression in insect cells. The DNAsegment can be present in the vector operably linked to regulatoryelements, for example, a promoter (e.g., T7, metallothionein I, orpolyhedrin promoters).

Polynucleotide sequences encoding GDF-16 can be expressed in eitherprokaryotes or eukaryotes. Hosts can include microbial, yeast, insectand mammalian organisms. Methods of expressing DNA sequences havingeukaryotic or viral sequences in prokaryotes are well known in the art.Biologically functional viral and plasmid DNA vectors capable ofexpression and replication in a host are known in the art. Such vectorsare used to incorporate DNA sequences of the invention. Preferably, themature C-terminal region of GDF-16 is expressed from a DNA clonecontaining the entire coding sequence of GDF-16. Alternatively, theC-terminal portion of GDF-16 can be expressed as a fusion protein withthe pro- region of another member of the TGF-β family or co-expressedwith another pro- region (see for example, Hammonds, et al., Molec.Endocrin. 5:149,1991; Gray, A., and Mason, A., Science, 247:1328, 1990).

Transformation of a host cell with recombinant DNA may be carried out byconventional techniques as are well known to those skilled in the art.Where the host is prokaryotic, such as E. coli, competent cells whichare capable of DNA uptake can be prepared from cells harvested afterexponential growth phase and subsequently treated by the CaCl₂ methodusing procedures well known in the art. Alternatively, MgCl₂, or RbClcan be used. Transformation can also be performed after forming aprotoplast of the host cell if desired.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate co-precipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with DNA sequences encoding the GDF-16 of the invention,and a second foreign DNA molecule encoding a selectable phenotype, suchas the herpes simplex thymidine kinase gene. Another method is to use aeukaryotic viral vector, such as simian virus 40 (SV40) or bovinepapilloma virus, to transiently infect or transform eukaryotic cells andexpress the protein, (see for example, Eukaryotic Viral Vectors, ColdSpring Harbor Laboratory, Gluzman ed., 1982).

Isolation and purification of microbial expressed polypeptide, orfragments thereof, provided by the invention, may be carried out byconventional means including preparative chromatography andimmunological separations involving monoclonal or polyclonal antibodies.

The GDF-16 polypeptides of the invention can also be used to produceantibodies which are immunoreactive or bind to epitopes of the GDF-16polypeptides. Antibody which consists essentially of pooled monoclonalantibodies with different epitopic specificities, as well as distinctmonoclonal antibody preparations are provided. Monoclonal antibodies aremade from antigen containing fragments of the protein by methods wellknown in the art (Kohler, et al., Nature, 256:495. 1975; CurrentProtocols in Molecular Biology, Ausubel, et al., ed., 1989).

The term “antibody” as used in this invention includes intact moleculesas well as fragments thereof, such as Fab, F(ab′)₂, and Fv which arecapable of binding the epitopic determinant. These antibody fragmentsretain some ability to selectively bind with its antigen or receptor andare defined as follows:

-   -   (1) Fab, the fragment which contains a monovalent        antigen-binding fragment of an antibody molecule can be produced        by digestion of whole antibody with the enzyme papain to yield        an intact light chain and a portion of one heavy chain;    -   (2) Fab′, the fragment of an antibody molecule can be obtained        by treating whole antibody with pepsin, followed by reduction,        to yield an intact light chain and a portion of the heavy chain;        two Fab′ fragments are obtained per antibody molecule;    -   (3) (Fab′)₂, the fragment of the antibody that can be obtained        by treating whole antibody with the enzyme pepsin without        subsequent reduction; F(ab′)₂ is a dimer of two Fab′ fragments        held together by two disulfide bonds;    -   (4) Fv, defined as a genetically engineered fragment containing        the variable region of the light chain and the variable region        of the heavy chain expressed as two chains; and    -   (5) Single chain antibody (“SCA”), defined as a genetically        engineered molecule containing the variable region of the light        chain, the variable region of the heavy chain, linked by a        suitable polypeptide linker as a genetically fused single chain        molecule.

Methods of making these fragments are known in the art. (See forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York (1988), incorporated herein by reference).

As used in this invention, the term “epitope” means any antigenicdeterminant on an antigen to which the paratope of an antibody binds.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics.

Antibodies which bind to the GDF-16 polypeptide of the invention can beprepared using an intact polypeptide or fragments containing smallpeptides of interest as the immunizing antigen. The polypeptide or apeptide used to immunize an animal can be derived from translated cDNAor chemical synthesis which can be conjugated to a carrier protein, ifdesired. Such commonly used carriers which are chemically coupled to thepeptide include keyhole limpet hemocyanin (KLH), thyroglobulin, bovineserum albumin (BSA), and tetanus toxoid. The coupled peptide is thenused to immunize the animal (e.g., a mouse, a rat, or a rabbit).

If desired, polyclonal or monoclonal antibodies can be further purified,for example, by binding to and elution from a matrix to which thepolypeptide or a peptide to which the antibodies were raised is bound.Those of skill in the art will know of various techniques common in theimmunology arts for purification and/or concentration of polyclonalantibodies, as well as monoclonal antibodies. (See for example, Coligan,et al., Unit 9, Current Protocols in Immunology, Wiley Interscience,1991 incorporated by reference).

It is also possible to use the anti-idiotype technology to producemonoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region which is the“image” of the epitope bound by the first monoclonal antibody.

The term “cell-proliferative disorder” denotes malignant as well asnon-malignant cell populations which often appear to differ from thesurrounding tissue both morphologically and genotypically. Malignantcells (i.e. cancer) develop as a result of a multistep process. TheGDF-16 polynucleotide that is an antisense molecule is useful intreating malignancies of the various organ systems. Essentially, anydisorder which is etiologically linked to altered expression of GDF-16could be considered susceptible to treatment with a GDF-16 suppressingreagent. One such disorder is a malignant cell proliferative disorder,for example.

The invention provides a method for detecting a cell proliferativedisorder which comprises contacting an anti-GDF-16 antibody with a cellsuspected of having a GDF-16 associated disorder and detecting bindingto the antibody. The antibody reactive with GDF-16 is labeled with acompound which allows detection of binding to GDF-16. For purposes ofthe invention, an antibody specific for GDF-16 polypeptide may be usedto detect the level of GDF-16 in biological fluids and tissues. Anyspecimen containing a detectable amount of antigen can be used. Thelevel of GDF-16 in the suspect cell can be compared with the level in anormal cell to determine whether the subject has a GDF-16-associatedcell proliferative disorder. Preferably the subject is human.

The antibodies of the invention can be used in any subject in which itis desirable to administer in vitro or in vivo immunodiagnosis orimmunotherapy. The antibodies of the invention are suited for use, forexample, in immunoassays in which they can be utilized in liquid phaseor bound to a solid phase carrier. In addition, the antibodies in theseimmunoassays can be detectably labeled in various ways. Examples oftypes of immunoassays which can utilize antibodies of the invention arecompetitive and non-competitive immunoassays in either a direct orindirect format. Examples of such immunoassays are the radioimmunoassay(RIA) and the sandwich (immunometric) assay. Detection of the antigensusing the antibodies of the invention can be done utilizing immunoassayswhich are run in either the forward, reverse, or simultaneous modes,including immunohistochemical assays on physiological samples. Those ofskill in the art will know, or can readily discern, other immunoassayformats without undue experimentation.

The antibodies of the invention can be bound to many different carriersand used to detect the presence of an antigen comprising the polypeptideof the invention. Examples of well-known carriers include glass,polystyrene, polypropylene, polyethylene, dextran, nylon, amylases,natural and modified celluloses, polyacrylamides, agaroses andmagnetite. The nature of the carrier can be either soluble or insolublefor purposes of the invention. Those skilled in the art will know ofother suitable carriers for binding antibodies, or will be able toascertain such, using routine experimentation.

There are many different labels and methods of labeling known to thoseof ordinary skill in the art. Examples of the types of labels which canbe used in the present invention include enzymes, radioisotopes,fluorescent compounds, colloidal metals, chemiluminescent compounds,phosphorescent compounds, and bioluminescent compounds. Those ofordinary skill in the art will know of other suitable labels for bindingto the antibody, or will be able to ascertain such, using routineexperimentation.

Another technique which may also result in greater sensitivity consistsof coupling the antibodies to low molecular weight haptens. Thesehaptens can then be specifically detected by means of a second reaction.For example, it is common to use such haptens as biotin, which reactswith avidin, or dinitrophenyl, puridoxal, and fluorescein, which canreact with specific antihapten antibodies.

In using the monoclonal antibodies of the invention for the in vivodetection of antigen, the detectably labeled antibody is given a dosewhich is diagnostically effective. The term “diagnostically effective”means that the amount of detectably labeled monoclonal antibody isadministered in sufficient quantity to enable detection of the sitehaving the antigen comprising a polypeptide of the invention for whichthe monoclonal antibodies are specific.

The concentration of detectably labeled monoclonal antibody which isadministered should be sufficient such that the binding to those cellshaving the polypeptide is detectable compared to the background.Further, it is desirable that the detectably labeled monoclonal antibodybe rapidly cleared from the circulatory system in order to give the besttarget-to-background signal ratio.

As a rule, the dosage of detectably labeled monoclonal antibody for invivo diagnosis will vary depending on such factors as age, sex, andextent of disease of the individual. Such dosages may vary, for example,depending on whether multiple injections are given, antigenic burden,and other factors known to those of skill in the art.

For in vivo diagnostic imaging, the type of detection instrumentavailable is a major factor in selecting a given radioisotope. Theradioisotope chosen must have a type of decay which is detectable for agiven type of instrument. Still another important factor in selecting aradioisotope for in vivo diagnosis is that deleterious radiation withrespect to the host is minimized. Ideally, a radioisotope used for invivo imaging will lack a particle emission, but produce a large numberof photons in the 140-250 keV range, which may readily be detected byconventional gamma cameras.

For in vivo diagnosis radioisotopes may be bound to immunoglobulineither directly or indirectly by using an intermediate functional group.Intermediate functional groups which often are used to bindradioisotopes which exist as metallic ions to immunoglobulins are thebifunctional chelating agents such as diethylenetriaminepentacetic acid(DTPA) and ethylenediaminetetraacetic acid (EDTA) and similar molecules.Typical examples of metallic ions which can be bound to the monoclonalantibodies of the invention are ¹¹¹In, ⁹⁷Ru, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ⁸⁹Zr, and²⁰¹T1.

The monoclonal antibodies of the invention can also be labeled with aparamagnetic isotope for purposes of in vivo diagnosis, as in magneticresonance imaging (MRI) or electron spin resonance (ESR). In general,any conventional method for visualizing diagnostic imaging can beutilized. Usually gamma and positron emitting radioisotopes are used forcamera imaging and paramagnetic isotopes for MRI. Elements which areparticularly useful in such techniques include ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr,and ⁵⁶Fe.

The monoclonal antibodies of the invention can be used in vitro and invivo to monitor the course of amelioration of a GDF-16-associateddisease in a subject. Thus, for example, by measuring the increase ordecrease in the number of cells expressing antigen comprising apolypeptide of the invention or changes in the concentration of suchantigen present in various body fluids, it would be possible todetermine whether a particular therapeutic regimen aimed at amelioratingthe GDF-16-associated disease is effective.

The term “ameliorate” denotes a lessening of the detrimental effect ofthe GDF-16-associated disease in the subject receiving therapy.

The present invention identifies a nucleotide sequence that can beexpressed in an altered manner as compared to expression in a normalcell, therefore it is possible to design appropriate therapeutic ordiagnostic techniques directed to this sequence. Thus, where acell-proliferative disorder is associated with the expression of GDF-16,nucleic acid sequences that interfere with GDF-16 expression at thetranslational level can be used. This approach utilizes, for example,antisense nucleic acid and ribozymes to block translation of a specificGDF-16 mRNA, either by masking that mRNA with an antisense nucleic acidor by cleaving it with a ribozyme.

Antisense nucleic acids are DNA or RNA molecules that are complementaryto at least a portion of a specific mRNA molecule (Weintraub. ScientificAmerican, 262:40, 1990). In the cell, the antisense nucleic acidshybridize to the corresponding mRNA, forming a double-stranded molecule.The antisense nucleic acids interfere with the translation of the mRNA,since the cell will not translate a mRNA that is double-stranded.Antisense oligomers of about 15 nucleotides are preferred, since theyare easily synthesized and are less likely to cause problems than largermolecules when introduced into the target GDF-16-producing cell. The useof antisense methods to inhibit the in vitro translation of genes iswell known in the art (Marcus-Sakura, Anal. Biochem., 172:289, 1988).

Ribozymes are RNA molecules possessing the ability to specificallycleave other single-stranded RNA in a manner analogous to DNArestriction endonucleases. Through the modification of nucleotidesequences which encode these RNAs, it is possible to engineer moleculesthat recognize specific nucleotide sequences in an RNA molecule andcleave it (Cech, J. Amer. Med. Assn., 260:3030, 1988). A major advantageof this approach is that, because they are sequence-specific, only mRNAswith particular sequences are inactivated.

There are two basic types of ribozymes namely, tetrahymena-type(Hasselhoff, Nature, 334:585, 1988) and “hammerhead” -type.Tetrahymena-type ribozymes recognize sequences which are four bases inlength, while “hammerhead” -type ribozymes recognize base sequences11-18 bases in length. The longer the recognition sequence, the greaterthe likelihood that the sequence will occur exclusively in the targetmRNA species. Consequently, hammerhead-type ribozymes are preferable totetrahymena-type ribozymes for inactivating a specific mRNA species and18-based recognition sequences are preferable to shorter recognitionsequences.

The present invention also provides gene therapy for the treatment ofcell proliferative or immunologic disorders which are mediated by GDF-16protein. Such therapy would achieve its therapeutic effect byintroduction of the GDF-16 antisense polynucleotide into cells havingthe proliferative disorder. Delivery of antisense GDF-16 polynucleotidecan be achieved using a recombinant expression vector such as a chimericvirus or a colloidal dispersion system. Especially preferred fortherapeutic delivery of antisense sequences is the use of targetedliposomes.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or, preferably, anRNA virus such as a retrovirus. Preferably, the retroviral vector is aderivative of a murine or avian retrovirus. Examples of retroviralvectors in which a single foreign gene can be inserted include, but arenot limited to: Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and RousSarcoma Virus (RSV). A number of additional retroviral vectors canincorporate multiple genes. All of these vectors can transfer orincorporate a gene for a selectable marker so that transduced cells canbe identified and generated. By inserting a GDF-16 sequence of interestinto the viral vector, along with another gene which encodes the ligandfor a receptor on a specific target cell, for example, the vector is nowtarget specific. Retroviral vectors can be made target specific byattaching, for example, a sugar, a glycolipid, or a protein. Preferredtargeting is accomplished by using an antibody to target the retroviralvector.

Those of skill in the art will know of, or can readily ascertain withoutundue experimentation, specific polynucleotide sequences which can beinserted into the retroviral genome or attached to a viral envelope toallow target specific delivery of the retroviral vector containing theGDF-16 antisense polynucleotide.

Since recombinant retroviruses are defective, they require assistance inorder to produce infectious vector particles. This assistance can beprovided, for example, by using helper cell lines that contain plasmidsencoding all of the structural genes of the retrovirus under the controlof regulatory sequences within the LTR. These plasmids are missing anucleotide sequence which enables the packaging mechanism to recognizean RNA transcript for encapsidation. Helper cell lines which havedeletions of the packaging signal include, but are not limited to Ψ2,PA317 and PA12, for example. These cell lines produce empty virions,since no genome is packaged. If a retroviral vector is introduced intosuch cells in which the packaging signal is intact, but the structuralgenes are replaced by other genes of interest, the vector can bepackaged and vector virion produced.

Alternatively, NIH 3T3 or other tissue culture cells can be directlytransfected with plasmids encoding the retroviral structural genes gag,pol and env, by conventional calcium phosphate transfection. These cellsare then transfected with the vector plasmid containing the genes ofinterest. The resulting cells release the retroviral vector into theculture medium.

Another targeted delivery system for GDF-16 antisense polynucleotides isa colloidal dispersion system. Colloidal dispersion systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. The preferred colloidal system of thisinvention is a liposome. Liposomes are artificial membrane vesicleswhich are useful as delivery vehicles in vitro and in vivo. It has beenshown that large unilamellar vesicles (LUV), which range in size from0.2-4.0 μm can encapsulate a substantial percentage of an aqueous buffercontaining large macromolecules. RNA, DNA and intact virions can beencapsulated within the aqueous interior and be delivered to cells in abiologically active form (Fraley, et al., Trends Biochem. Sci., 6:77,1981). In addition to mammalian cells, liposomes have been used fordelivery of polynucleotides in plant, yeast and bacterial cells. Inorder for a liposome to be an efficient gene transfer vehicle, thefollowing characteristics should be present: (1) encapsulation of thegenes of interest at high efficiency while not compromising theirbiological activity; (2) preferential and substantial binding to atarget cell in comparison to non-target cells; (3) delivery of theaqueous contents of the vesicle to the target cell cytoplasm at highefficiency; and (4) accurate and effective expression of geneticinformation (Mannino, et al., Biotechniques, 6:682, 1988).

The composition of the liposome is usually a combination ofphospholipids, particularly high-phase-transition-temperaturephospholipids, usually in combination with steroids, especiallycholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidyletha-nolamine, sphingolipids,cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphos-phatidylcholine.

The targeting of liposomes can be classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticuloendothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

The surface of the targeted delivery system may be modified in a varietyof ways. In the case of a liposomal targeted delivery system, lipidgroups can be incorporated into the lipid bilayer of the liposome inorder to maintain the targeting ligand in stable association with theliposomal bilayer. Various linking groups can be used for joining thelipid chains to the targeting ligand.

In another embodiment, the invention provides a method for identifyingGDF-16 receptor polypeptide comprising incubating components comprisingGDF-16 polypeptide and a cell expressing a receptor or a solublereceptor under conditions sufficient to allow the GDF to bind to thereceptor; measuring the binding of the GDF polypeptide to the receptor;and isolating the receptor. Methods of isolating the receptors aredescribed in more detail in the Examples section.

In yet another embodiment, the present invention relates to GDF-16receptor-binding agents that block binding of GDF-16 to their receptors.Such agents could represent research and diagnostic tools in the studyof muscle wasting disorder as described above and the development ofmore effective therapeutics. In addition, pharmaceutical compositionscomprising GDF-16 receptor-binding agents may represent effectivetherapeutics. In the context of the invention, the phrase “GDF-16receptor-binding agent” denotes a naturally occurring ligand of GDF-16receptors such as, for example, GDF-16, a synthetic ligand of GDF-16receptors, or appropriate derivatives of the natural or syntheticligands. The determination and isolation of ligands is well described inthe art. See, e.g., Lemer, Trends NeuroSci. 17:142-146 (1994) which ishereby incorporated in its entirety by reference.

In yet another embodiment, the present invention relates to GDF-16receptor-binding agents that interfere with binding between GDF-16receptor and a GDF-16. Such binding agents may interfere by competitiveinhibition, by non-competitive inhibition or by uncompetitiveinhibition. Interference with normal binding between GDF-16 receptorsand one or more GDF-16 can result in a useful pharmacological effect.

In another embodiment, the invention provides a method for identifying acomposition which binds to GDF-16 receptors. The method includesincubating components comprising the composition and GDF-16 receptorsunder conditions sufficient to allow the components to interact andmeasuring the binding of the composition to GDF-16 receptors.Compositions that bind to GDF-16 receptors include peptides,peptidomimetics, polypeptides, chemical compounds and biologic agents asdescribed above.

Incubating includes conditions which allow contact between the testcomposition and GDF-16 receptors. Contacting includes in solution and insolid phase. The test ligand(s)/composition may optionally be acombinatorial library for screening a plurality of compositions.Compositions identified in the method of the invention can be furtherevaluated, detected, cloned, sequenced, and the like, either in solutionor after binding to a solid support, by any method usually applied tothe detection of a specific DNA sequence such as PCR, oligomerrestriction (Saiki, et al., Bio/Technology, 3:1008-1012, 1985),allele-specific oligonucleotide (ASO) probe analysis (Conner, et al.,Proc. Natl. Acad. Sci. USA, 80:278, 1983), oligonucleotide ligationassays (OLAs) (Landegren, et al., Science, 241:1077, 1988), and thelike. Molecular techniques for DNA analysis have been reviewed(Landegren, et al., Science, 242:229-237, 1988).

To determine if a composition can functionally complex with the receptorprotein, induction of the exogenous gene is monitored by monitoringchanges in the protein levels of the protein encoded for by theexogenous gene, for example. When a composition(s) is found that caninduce transcription of the exogenous gene, it is concluded that thiscomposition(s) can bind to the receptor protein coded for by the nucleicacid encoding the initial sample test composition(s).

Expression of the exogenous gene can be monitored by a functional assayor assay for a protein product, for example. The exogenous gene istherefore a gene which will provide an assayable/measurable expressionproduct in order to allow detection of expression of the exogenous gene.Such exogenous genes include, but are not limited to, reporter genessuch as chloramphenicol acetyltransferase gene, an alkaline phosphatasegene, beta-galactosidase,a luciferase gene, a green fluorescent proteingene, guanine xanthine phosphoribosyltransferase, alkaline phosphatase,and antibiotic resistance genes (e.g., neomycin phosphotransferase).

Expression of the exogenous gene is indicative of composition-receptorbinding, thus, the binding or blocking composition can be identified andisolated. The compositions of the present invention can be extracted andpurified from the culture media or a cell by using known proteinpurification techniques commonly employed, such as extraction,precipitation, ion exchange chromatography, affinity chromatography, gelfiltration and the like. Compositions can be isolated by affinitychromatography using the modified receptor protein extracellular domainbound to a column matrix or by heparin chromatography.

Also included in the screening method of the invention is combinatorialchemistry methods for identifying chemical compounds that bind to GDF-16receptors. Thus, the screening method is also useful for identifyingvariants, binding or blocking agents, etc., which functionally, if notphysically (e.g., sterically) act as antagonists or agonists, asdesired.

There are a variety of applications using the polypeptide,polynucleotide, and antibodies of the invention including treatment ofcell proliferative and immunologic disorders. In addition, GDF-16 may beuseful in various gene therapy procedures.

The following examples are intended to illustrate but not limit theinvention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may alternatively be used.

EXAMPLE 1 Identification and Isolation of a Novel TGF-β Family Member

GDF-16 was isolated from human genomic libraries using murine lefty as aprobe. Hybridization was carried out in 1.08 M NaCl, 60 mM sodiumphosphate, 6 mM EDTA, 48 mM NaOH, 0.5% SDS, 0.2% bovine serum albumin,0.2% ficoll, and 0.2% polyvinyl pyrrolidone at 65° C. Final washes werecarried out in 0.3 M NaCl, 30 mM sodium citrate at 58° C.

The sequence of GDF-16 deduced from analysis of genomic clones is shownin FIG. 1. The sequence contains an open reading frame encoding aprotein showing significant homology to know members of the TGF-βsuperfamily. The sequence contains a putative RXXR proteolyticprocessing site that is followed by a sequence of 101 amino acids thatcontains all of the hallmarks of the TGF-β superfamily. By analogy withknown family members, a dimer of the C-terminal 101 amino acids ispredicted to represent the active GDF-16 molecule.

EXAMPLE 2 Expression of GDF-16

To determine the expression pattern of GDF-16, a Northern blotcontaining RNA samples prepared from a variety of human tissue(Clontech) is probed with GDF-16. Northern analysis is carried out asdescribed previously (Lee, Mol. Endocrinol., 4:1034, 1990) except thatthe hybridization is carried out in 5×SSPE, 10% dextran sulfate, 50%formamide, 1% SDS, 200 μg/ml salmon DNA, and 0.1% each of bovine serumalbumin, ficoll, and polyvinylpyrrolidone.

Although the invention has been described with reference to thepresently preferred embodiment, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

1. Substantially pure growth differentiation factor-16 (GDF-16). 2.Antibodies that bind to the polypeptide of claim 1 or fragments thereof.3. The antibodies of claim 2, wherein the antibodies are polyclonal. 4.The antibodies of claim 2, wherein the antibodies are monoclonal.
 5. Amethod of treating a cell proliferative disorder or immunologic disorderassociated with expression of GDF-16, comprising contacting the cellswith a reagent which suppresses the GDF-16 activity.
 6. The method ofclaim 5, wherein the reagent is an anti-GDF-16 antibody.
 7. The methodof claim 5, wherein the reagent is a GDF-16 antisense sequence.
 8. Themethod of claim 5, wherein the reagent which suppresses GDF-16 activityis introduced to a cell using a vector.
 9. The method of claim 8,wherein the vector is a colloidal dispersion system.
 10. The method ofclaim 9, wherein the colloidal dispersion system is a liposome.
 11. Themethod of claim 10, wherein the liposome is essentially target specific.12. The method of claim 11, wherein the liposome is anatomicallytargeted.
 13. The method of claim 12, wherein the liposome ismechanistically targeted.
 14. The method of claim 13, wherein themechanistic targeting is passive.
 15. The method of claim 13, whereinthe mechanistic targeting is active.
 16. The method of claim 15, whereinthe liposome is actively targeted by coupling with a moiety selectedfrom the group consisting of a sugar, a glycolipid, and a protein. 17.The method of claim 16, wherein the protein moiety is an antibody. 18.The method of claim 17, wherein the vector is a virus.
 19. The method ofclaim 18, wherein the virus is an RNA virus.
 20. The method of claim 19,wherein the RNA virus is a retrovirus.
 21. The method of claim 20,wherein the retrovirus is essentially target specific.
 22. The method ofclaim 21, wherein a moiety for target specificity is encoded by apolynucleotide inserted into the retroviral genome.
 23. The method ofclaim 21, wherein a moiety for target specificity is selected from thegroup consisting of a sugar, a glycolipid, and a protein.
 24. The methodof claim 23, wherein the protein is an antibody.
 25. A method foridentifying a GDF-16 receptor polypeptide comprising: a) incubatingcomponents comprising GDF-16 polypeptide and a cell expressing areceptor or a soluble receptor under conditions sufficient to allow theGDF-16 to bind to the receptor; b) measuring the binding of the GDF-16polypeptide to the receptor; and c) isolating the receptor.